Protocol

Fixation

Wash

Embryos were washed in 0.1M Cacodylate Buffer + 0.1M Sucrose for 2x10 mins. Embryos for storage were kept in this solution at 4oC.

Post-fixation

If post-fixation was to be carried out, embryos were washed in 0.1M Cacodylate Buffer without sucrose for 2x10 mins before fixing in 1% Osmium Tetroxide in 0.1M Cacodylate Buffer for 30-60 mins depending on age.

Wash

Embryos were washed in 0.1M Cacodylate Buffer for 2x10mins.

Dehydration

Increasing concentrations of ethanol in distilled water were used to dehydrate the embryos. 10%,30%,50%,70%90% ethanol each for 2x10mins 0-5day and 2x30mins for 6-8 day before a final 3 changes of 100% dry ethanol.

Infiltration

Due to the very fragile nature of these young embryos, Propylene Oxide was omitted as a transition fluid for the Araldite resin. Instead mixtures of Araldite plus accelerator (DMP30) in 'dry' ethanol (1:2, 1:1 and 2:1 for 2 hrs each) were used to assist the infiltration of 6-8 day embryos. The young 0-5 day embryos were given a 1:1 Araldite/ethanol mix for 2hrs.
All embryos were then transferred into a thin layer of complete resin overnight at room temperature to infiltrate.
At the same time a thin layer of Araldite plus accelerator was poured onto
the bottom of the final embedding moulds ( flat aluminium foil dishes) and polymerised overnight at 60oC to give the bottom layer of the 'sandwich' technique used for embedding.

Embedding

After the infiltration stage was complete the embryos were transferred to fresh Araldite plus accelerator that had been added to the foil dishes as another thin layer. The embryos were allowed to sink to the interface of the two layers, carefully orientated with a fine glass rod, then placed in a 60oc oven for 3 days to polymerise.